Urotensin II (U-II) is a cyclic peptide that was originally isolated from the goby Gillichthys mirabilis (H. A. Bern et al., Recent Prog. Horm. Res., 1995, 41, 533-552). U-II was found to cause smooth muscle and vascular constriction in fish as well as having effects on osmoregulation. Goby U-II was found to have variable effects on mammalian smooth muscle. For example, both vasorelaxation and vasoconstriction were observed in isolated rat aorta (A. Gibson, Br. J. Pharmacol., 1987, 91, 205-212). Subsequently, U-II was cloned from other species including man (Y. Coulouarn et al., Proc. Nat. Acad. Sci., USA, 1998, 95, 15803-15808). It was found that U-II from several species, including mammalian and non-mammalian species, contains a conserved hexapeptide core sequence although there is some variation at the amino terminus (J. M. Conlon et al., J. Exp. Zool., 1996, 275, 226-238).
The human receptor for U-II, a G Protein-coupled receptor homologous to rat GPR14 was identified in 1999 (R. S. Ames et al., Nature, 1999, 401, 282-286). The receptor (UT receptor) was found to be expressed predominantly in the cardiorenal system, although it is present in other tissues as well including skeletal muscle, adrenal gland, pancreas, thyroid, lung, liver, testis, and nervous system tissue. In blood vessels, the UT receptor was found to be expressed in cardiac and arterial tissue but not in venous tissue. Co-expression of U-II and the UT receptor in cardiovascular tissues was demonstrated in a 2001 study (M. Matsushita et al., J. Hypertens., 2001, 19, 2185-2190).
U-II was found to be a potent vasoconstrictor in isolated arteries from rats (R. S. Ames et al., 1999, ibid), humans (J. J. Maguire et al., Br. J. Pharmacol., 131, 441-446) and other species (S. A. Douglas et al., Br. J. Pharmacol., 2000, 131, 1262-1274). Consistent with the UT receptor expression pattern noted above, with few exceptions, the vasoconstrictor activity of U-II is restricted to the arterial portion of the vasculature. Evidence for a specific U-II/UT receptor interaction being responsible for the vasoconstrictor response to U-II was provided in a 2003 study which showed that the response was eliminated in blood vessels from UT receptor knock-out mice (D. J. Behm et al., Br. J. Pharmacol., 2003, 139, 464-472).
Systemic effects of U-II can vary depending on the species and method of administration. In one study, in rats, i.v. bolus administration of U-II resulted in a dose-dependent decrease of blood pressure and cardiac contractility (G. S. Hassan et al., Can. J. Physiol. Pharmacol., 2003, 81, 125-128). However, a later study showed that slow, i.v. infusion of U-II in the rat increased systemic arterial blood pressure and splanchnic vascular resistance (T. Bennet et al., Br. J. Pharmacol., 2002, 135, 200). In a study with anesthetized monkeys, systemic administration of human U-II resulted in severe myocardial depression and fatal circulatory collapse. At a 300 pmol/kg dose, peripheral resistance increased by 300% without inducing systemic hypertension (R. S. Ames et al., 1999, ibid). In healthy, human volunteers, intradermal injection of U-II resulted in a dose-dependant decrease of blood flow with sustained vasoconstriction (S. J. Leslie et al., Circulation, 2001, 102, Suppl. II, 542).
In addition to the cardiovascular effects noted above, U-II was found to have mitogenic and hypertrophic effects. In vascular smooth muscle cells from rabbit aorta, U-II was found to increase cell proliferation (V. Sauzeau et al., Circ. Res., 2001, 88, 1102-1104) through activation of Rho A and Rho kinase. In addition, U-II was found to induce cellular hypertrophy in rat cardiomyocytes (Y. Zou et al., FEBS Lett., 2001, 508, 57-60). Cellular proliferation and hypertrophy in the vasculature are associated with remodeling of blood vessel walls and myocardial tissue which occur in hypertension and heart failure.
Studies such as the ones cited above indicate that U-II has complex effects on the vasculature of the cardiorenal system and therefore may play a role in cardiovascular tone, structure and disease. In fact, clinical measurement of tissue expression or circulating and urinary levels of U-II have found up-regulation of U-II or its receptor in several cardiorenal diseases. In one study, urinary U-II concentrations in patients with hypertension or renal tubular abnormality were significantly higher than those found in normal individuals (M. Matsushita et al., 2001, ibid). In another study, U-II was found to be up-regulated in cardiomyocytes, endothelial cells and vascular smooth muscle cells of damaged heart tissue from end-stage congestive heart failure patients compared to controls. In addition, the UT receptor exhibited greater binding density in diseased tissue compared to normal (S. A. Douglas et al., Lancet, 2002, 359, 1990-1997). Furthermore, plasma levels of U-II were also found to be higher in heart failure patients than in normal controls (A. M. Richards et al., Lancet, 2002, 360, 545-546). Increased levels of U-II were also observed in patients with diabetes mellitus (K. Totsune et al., Clin. Sci., 2003, 104, 1-5) and portal hypertension and cirrhosis (J. Heller et al., J. Hepatol., 2002, 37, 767-772). The studies cited above suggest a possible role for U-II in these diseases.
In response to the physiological profile and its possible role in disease, reports of a variety of U-II receptor antagonists have appeared (see for example S. A. Douglas et al., Trends in Pharmacological Sciences, 2004, 25, 76-85; D. Dhanak et al., Ann Rep. Med. Chem., 2003, 38, 99-110). Two recent publications on the U-II receptor antagonist palosuran reported that the antagonist was able to improve survival, increase insulin, slow the increase of glycemia and delay renal damage in a streptozotocin-induced diabetic rat model (M. Clozel et al., J. Pharmacol. Exp. Ther., 2006, 316, 1115-1121) and was effective in a rat model of renal ischemia, preventing both post-ischemic renal vasoconstriction and acute renal failure (M. Clozel et al., J. Pharmacol. Exp. Ther. 2006, 316, 1115-1121 (P. Sidharta et al., Clin. Pharmacol. Ther. 2006, 80, 246-56.)). Furthermore, in a clinical trial the UT antagonist Palosuran was found to modulate the 24-hour urinary albumin excretion rate in patients by 24.3%. These studies further validate inhibition of U-II as a therapeutic target.
There remains an unmet medical need for new drugs to treat cardiovascular disease. A study published in 2003 estimated that almost 29% of the adult U.S. population had hypertension in 1999-2000 (I. Hajjar et al., JAMA, 2003, 290, 199-206). Furthermore, 69% of the hypertensive individuals studied during this period did not have their hypertension controlled at the time their blood pressure was measured. This figure was worse in patients with diabetes and hypertension where 75% of those patients studied did not have their blood pressure controlled to the target level. Another more recent study showed similar results, with less than one-third of hypertensive patients studied having blood pressure controlled to the target level (V. Andros, Am. J. Manag. Care, 2005, 11, S215-S219). Therefore, despite the number of medications available to treat hypertension, including diuretics, beta blockers, angiotensin converting enzyme inhibitors, angiotensin blockers and calcium channel blockers, hypertension remains poorly controlled or resistant to current medication for many patients. If not adequately treated, hypertension can lead to other cardiovascular diseases and organ failure including coronary artery disease, stroke, myocardial infarction, heart failure, renal failure, and peripheral artery disease.
U-II and its receptor are coexpressed in the heart and are upregulated during cardiac dysfunction. Although the expression of UT is low to undetectable in the healthy myocardium, U-II and its receptor are up-regulated in patients with moderate and end-stage heart failure. In addition, expression of UT is up-regulated in the ischemic, chronic hypoxic and post-myocardial infracted rat myocardium. Interestingly, UT up-regulation in the myocardium of hypoxic rats is accompanied by sustained ventricular hypertrophy. In vivo, short-term hypertrophic growth of cardiomyocytes is an adaptive response that increases cardiac output in cardiac dysfunction (heart failure, hypertension) and injury (myocardial infarction), whereas sustained hypertrophic growth is maladaptive and can cause adverse cardiac remodeling, which is characterized by inflammation, fibrosis, and cardiac ventricular hypertrophy. Furthermore, a UT inhibitor was found to be efficacious in a rat CHF model (N. Bousette et al., J. Mol. Cell. Cardiol. 2006, 41, 5-95; N. Bousette et al., Peptides 2006, 27, 19-2926). This study concluded that treatment with the UT antagonist provided significant reduction of overall mortality, left ventricular end-diastolic pressure, lung edema, right ventricular systolic pressure, central venous pressure, cardiomyocyte hypertrophy, and ventricular dilatation. Therefore, an orally active small molecule UT antagonist may modulate the pathological changes that occur during CHF, end-organ damage, or hypertension.
Increased expression of U-II has been shown to be associated with atherosclerosis. In humans, U-II expression (immunohistochemical staining) is increased in atherosclerotic plaques (endothelial, smooth muscle and inflammatory cells of both carotid and aortic plaques) and the increased expression correlates with disease progression (N. Bousette, et al., Athero. 2004, 176, 117-123). In atherosclerosis rodent models (apoE−/− mice) there is an upregulation of mRNA urotensin receptor (but not U-II) in the atherosclerotic aorta (Z. Wang, et al., Peptides 2006, 27, 858-863). Several in vitro studies have demonstrated a functional role for U-II in atherosclerosis pathology. U-II may play a novel role in the formation of macrophage-derived foam cells by upregulating ACAT-1 expression via the UT receptor/G-protein/c-Src/PKC/MEK and ROCK pathways but not by SR-A, thus contributing to the relatively rapid development of atherosclerosis in hypertension (T. Wantanabe, et al., Hypertension 2005, 46, 738-744). U-II and 5-HT may induce the synergistic interaction in inducing VSMC proliferation via a G-protein-coupled receptor/PKC/Src tyrosine kinase/MAPK pathway, thus contributing to the relatively rapid development of atherosclerosis in hypertensive vascular disease (J. Hypertension 19, 2191-2196). U-II acts synergistically with moxLDL in inducing VSMC proliferation via the c-Src/PKC/MAPK pathway, which may explain the relatively rapid progression of atherosclerosis in patients with hypertension and hypercholesterolemia (T. Wantanabe, et al., Circulation 2001, 104, 16-18, 2001).